In life science and medical applications, there is a need for systematic analysis of disease-related proteins in connection with treatment and prevention of diseases. In particular, with the development of basic research for medicine discovery including molecular biology and genomics (genome science), regions for drug discovery have recently been subjected to a rapid change, and novel methods of drug discovery representative of genomic drug discovery have been developed.
As such, in life science and medical applications for developing novel pharmaceutical drugs, substances having physiological activity in specific diseases or environments must be checked. Most of the biological substances are composed of protein, and explanation of structure and function of the protein falls under basic issues in life science and medical applications. In particular, about 35,000 kinds of human genes have been identified, but the number of proteins produced by genomes is estimated to range from hundreds of thousands to several millions. Since the proteins are involved in all necessary reactions within cell organelle, it is necessary to systematically conduct research on functions of genes at the level of proteins.
Meanwhile, the proteins are very complicated due to various properties such as a molecular weight, isoelectric point (pI), hydrophilicity, hydrophobicity, and so on. Thus, in order to analyze the proteins, it is necessary to primarily separate the proteins, and then to indentify the separated proteins in connection with mass spectrometry, bioinformatics, and so on. In this process, disease-related proteins are relatively smaller than the other sound proteins, it is necessary to analyze the proteins using high-performance protein separation technology. In particular, the proteins are very important to life activities, and represent their functions in interaction with other molecules including protein molecules, DNA molecules, synthetic compounds or photons. For this reason, in order to understand specific proteins, which interaction occurs along with any molecule is examined by identifying molecules having an influence on each other and by explaining a mechanism of the interaction (physiological action), instead of being limited to merely specifying the physical and/or chemical properties of these molecules.
A typical method of separating and analyzing the proteins is sodiumdodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), in which the proteins are separated due to a difference in selectivity by applying an electric field to a polyacrylamide gel plate. This method is a one-dimensional separation method that is widely used in the field of separating and identifying proteins in order of molecular weight and in the process of separating and identifying simple proteins, and has a problem in that the proteins have a tertiary structure denatured in an SDS solution, or are confined in the gel.
Meanwhile, the proteins produced by a genome are genetically defined as a proteome. When many protein mixtures including the proteome, in which generation, relative abundance, etc. of the protein are determined depending on an intracellular position or the physiological state of a cell or an organ, are separated, and then properties of the protein mixtures are identified, the identification itself is impossible using the aforementioned one-dimensional separation technology alone. Thus, in order to separate the protein mixtures, a two-dimensional (2D) separation method called 2D-polyacrylamide gel electrophoresis (2D-PAGE) is used, in which the proteins, which are primarily separated according to the protein property, are secondarily separated according to the molecular weight of the protein [Zhou, F. et al. Anal. Chem. 2004, 76, pp. 2734-2740; Klose, J. et al. Electrophoresis, 1995, 16, pp. 1034-1059; Righetti, P. G.1 et al. “Prefractionation Techniques in Proteome Analysis,” Anal. Chem. 2001, 73, pp. 320-326]. In this 2D-PAGE, the proteins are subjected to isolectric focusing (IEF) in a narrow gel strip, i.e. an ampholyte carrier, in which a pH gradient is fixedly formed, according to isoelectric point (pI). This process requires about 12 hours or more.
Next, the proteins are separated in order of the pI. Subsequently, the gel strip is fixed on the upper end of a polyacrylamide gel plate in a transverse direction, and then the proteins are subjected to electrophoresis in a longitudinal direction. Thereby, the proteins are separated in the longitudinal direction according to the order of size, i.e. molecular weight. At this time, the proteins having low molecular weight mainly move to a lower end of the polyacrylamide gel plate. A total time required for the 2D-PAGE separation amounts to about 36 hours. After the 2D separation is terminated, protein spots shown on the polyacrylamide gel plate are dyed, thereby checking the number of proteins. If necessary, the protein of each spot is recovered, decomposed by an enzyme, and identified using mass spectroscopy.
This 2D-PAGE is very useful in checking a rough pattern of the proteins due to its high resolution. Further, the 2D-PAGE allows the proteins to be separated on a semi-preparative scale, so that it can be used for analysis of human plasma proteins having the shape of a very complicated protein mixture as well as proteins extracted from urine, various biologic tissues, etc., and clinical detection and diagnosis of diseases [Giddings, J. C., Unified Separation Science, John Wiley & Sons, New York 1991, pp. 126-128].
However, the 2D-PAGE is a labor-intensive method, is difficult to automate, and is limited in detection sensitivity and dynamic range. Further, separating the proteins without denaturation is difficult in the 2D-PAGE because the SDS solution is used to separate the proteins, which results in separation of the proteins in the denatured state, and recovering the sample is not easy because the separated proteins are also confined in a gel matrix. As such, the 2D-PAGE must usually decompose the proteins in the gel using the enzyme, and recover and analyze the proteins in a peptide form.
Meanwhile, a capillary isoelectric focusing (CIEF) method is a method involving filling ampholyte carriers in silica capillaries along with proteins, applying an electric field to separate the proteins according to pI of the protein [Conti, M. et al. Electrophoresis 1996, 17, pp. 1485-1491]. Here, in comparison with the IEF of the 2D-PAGE, the CIEF is the same in principle, but different in that it uses the silica capillaries rather than the gel strip. The CIEF can be used to process a small quantity of protein samples due to the intracapillary separation, and to separate the proteins having a slight difference of 0.003 between their pI values when it has high sensitivity [Quigley, W. C. et al. J. Anal. Chem. 2000, 76, pp. 4645-4658]. Further, despite the capability of processing the small quantity of protein samples due to the intracapillary separation and the high sensitivity, the CIEF is limited in its separation capability to process complicated protein mixtures such as proteome. In order to increase separation efficiency, an attempt has recently been made to use the CIEF in on-line connection with a secondary separation method such as chromatography rather than as a single analysis technique.
A typical example of the technology that carries out the 2D separation in on-line connection with the CIEF is CIEF-reversed phase liquid chromatography (RPLC) that connects the CIEF with the RPLC on line. The CIEF-RPLC secondarily separates proteins or peptide bands, which are separated by pI regions in the CIEF, in a chromatography column according to hydrophobicity difference between peptides [Chen, J. et al. Electrophoresis 2002, 23, pp. 3143-3148]. With the use of this method, a result of conducting a test with peptide mixtures obtained by hydrolyzing the proteome of a fruit fly, Drosophila melanogaster, was that a peak capacity of more than 1800 could be obtained through separation of about 8 hours.
Further, CIEF-capillary gel electrophoresis (CGE) connecting the CIEF with the CGE on line can be used to carry out separation in capillaries filled with a polyacrylamide gel instead of the aforementioned polyacrylamide gel plate according to molecular weight, and attempt to separate simple proteins such as hemoglobin [Yang, C.1 et al. Anal. Chem. 2003, 75, pp. 215-218]. The CIEF-CGE is useful in separating peptide mixtures, which are obtained by hydrolyzing proteins with protein enzymes, rather than the proteins. However, due to protein chain breakdown occurring when the proteins pass through the chromatography column, protein loss within the chromatography column, and so on, it is difficult to apply the CIEF-CGE to the proteins. The CIEF-RPLC may be connected on line with a mass spectrometer using electrospray ionization (ESI) [Tnag, Q. et al. Anal. Chem. 1996, 68, pp. 2482-2487; Yang, L. et al. Anal. Chem. 1998, 70, pp. 3235-3241; Martinovi, S. et al. Anal. Chem. 2000, 72, pp. 5356-5360]. However, because the ampholyte used for the CIEF separation is not removed, the CIEF-RPLC must be subjected to separate purification in order to remove the ampholyte after the CIEF separation. As such, sample analysis is difficult in the CIEF-RPLC due to inhibition of ions in the solution without previous removal of the ampholyte. In order to solve this problem, the ampholyte must be considerably removed using membranes such as microdialyzable cathode cells [Zhou, F. et al. Anal. Chem. 2004, 76, pp. 2734-2740]. However, although the CIEF-RPLC is used to separate the proteins, denaturation of the proteins cannot be avoided due to use of an organic solvent during the RPLC separation, and the CIEF-RPLC cannot be applied to the separation of the proteins having large molecular weight.
Meanwhile, an example of the method of separating the proteins according to the order of molecular weight is flow field-flow fractionation (FlFFF), which is a type of field flow fractionation (FFF). The FIFFF is a separation analysis technique that is used for size-based separation of proteins, cells, water-soluble polymers, and nano-particles, as well as property analysis of a diffusion coefficient, particle size, molecular weight, and so on etc. [J. C. Giddings, et al. Science 1976, 193, p. 1244; M. H. Moon, et al. Anal. Chem. 1999, 71(14), p. 2657]. In the FFF-based separation, an available channel is a channel that has a cuboidal cross section and a hollow space. Further, because there is no stationary phase, samples are separated depending on the strength of an external field applied in a direction perpendicular to the flow of a fluid moving the samples along a channel axis. Thus, the FIFFF uses a cross flow of the fluid, and controls retention of macro-molecules such as proteins by regulating a flow rate of the cross flow.
The retention of the samples in the FIFFF channel is caused by a balance between the flow rate of the cross flow flowing out through a channel bottom and Brownian diffusion of the samples. In the case of the proteins, an average height of the samples moving in the channel is determined by a degree of the Brownian diffusion which varies depending on molecular weight or a Stoke's diameter. The smaller the molecular weight, the greater the diffusion. Thus, the diffusion and the flow rate of the cross flow are balanced at a position where the proteins are distant from the channel bottom. At this time, a separation flow flowing along a channel axis is a parabolic shape, and the proteins and the macro-molecule samples are separated according to size. Accordingly, the samples having low molecular weight are discharged from the channel, so that the samples are separated according to the size of molecular weight.
FIG. 1 shows the configuration of a flow field-flow fractionation (FIFFF) system applied to separation of proteins. An available channel is called an AFIFFF(asymmetrical flow field-flow fractionation) channel. This channel has an asymmetrical channel structure in which only a lower block under a channel has a frit, unlike a conventional symmetrical channel structure in which upper and lower blocks of a channel have respective frits. A fluid is transferred from a high performance liquid chromatography (HPLC) pump, and protein samples separated and eluted in the channel are detected using an ultraviolet/visible radiation (UV/VIS) detector. The proteins are separated in the AFIFFF channel as follows. The protein samples injected into the channel through an injector are subjected to a sample relaxation-focusing process before the separation is initiated. This sample relaxation-focusing process serves to put the samples in equilibrium between the strength of an external field applied outside the samples and diffusion of the samples, and is an essential process for the AFIFFF. The sample relaxation-focusing is carried out by injecting the fluid through an inlet of the channel as well as an outlet of the channel or an inlet of a focusing flow as in FIG. 1 to adjust a ratio of flow rates of the two flows such that the samples injected into the channel can be focused at a position corresponding to a triangular base of the channel inlet. Usually, this sample relaxation-focusing is experimentally applied by computing a ratio of a position where the samples are subjected to relaxation and focusing to an entire length of the channel to calculate the flow rate ratio. For example, the flow rate ratio may be finally determined by injecting a material such as organic dye, water-soluble ink, or the like to check a position where the material is focused. The sample relaxation-focusing requires a sufficient time for a buffer solution corresponding to a volume of the channel to flow out through the channel bottom. When the sample relaxation-focusing process is completed, inflow of the focusing flow is interrupted, and the fluid is transferred only to the channel inlet. At this time, a ratio of a cross flow flowing out through the channel bottom to an outlet flow transferred to a detector is adjusted. Thereby, the proteins are separated. In the case where the focusing flow is adjusted in such a manner that a part thereof can flow into the detector during the sample relaxation-focusing and be transferred by the flow rate of the outlet flow used when the proteins are separated, a phenomenon in which the flow of the fluid comes to a standstill at the detector can be avoided when the relaxation-focusing process is transited to the separation process.
In the FIFFF using the asymmetrical channel, when the proteins are separated, the proteins can be separated in order from low molecular weight to high molecular weight. Because the separation solution uses the buffer solution, the proteins are separated without denaturation. Because no filler is filled in the channel, risks such as breakdown of the protein samples or blocking of the separation channel can be minimized. When thickness and width of a spacer determining the volume of the channel are adjusted, the flow rate of the fluid, separation efficiency, etc. can be varied, and the proteins can be separated at a micro flow rate, which is suitable to separate a very small amount of proteins [D. Kang, M. H. Moon, Anal. Chem. 2004, 76, pp. 3851-3855; S. Oh et al. J. Separation Sci., 2007, 30, pp. 1982-1087].
In comparison with the gel electrophoresis, the separation method using the AFIFFF can be used to separate the proteins in order from low molecular weight to high molecular weight, and minimize the breakdown of the protein samples or the blocking of the separation channel. However, the separation method using the AFIFFF is not very high in separation capability, and has difficulty in carrying out the separation on the basis of various properties of the protein.
As a plan to overcome this problem, a 2D separation method, CIEF-Hollow fiber flow-field flow fractionation (HF FIFFF) has been developed, whereby isoelectric focusing and molecular weight based separation without using the gel are possible. The CIEF-HF FIFFF is configured to serially connect HF FIFFF with the CIEF method that carries out the isoelectric focusing in the capillaries, and more particularly, fills the ampholyte carriers in the silica capillaries along with the proteins, and applies the electric field to separate the proteins according to the pI of the protein [Conti, M.; Gelfi, C.; Righetti, P. G. Electrophoresis 1996, 17, pp. 1485-1491]. The HF FIFFF belongs to another example of separating and analyzing the proteins, and is a separation method that uses a hollow fiber membrane as a separation channel [W. J. Lee, B.-R. Min, M. H. Moon, Anal. Chem. 1999, 71(16), p. 3446; M. H. Moon, K. H. Lee, B.-R. Min, J. Microcolumn September, 1999, 11(9), p. 676; P. Reschiglian, et al. Anal. Chem. 2005, 77, p. 47]. In the HF FIFFF, the function of an external field is determined by the flow rate of a cross flow or a radial flow discharged to an outer wall of the hollow fiber membrane, and samples in the channel maintain an equilibrium with the external field. In this case, as shown in FIG. 2, the samples proceed in the shape of a circular band. At this time, a ratio of the flow of the samples to a separation flow moving toward a longitudinal axis of the channel is adjusted, thereby adjusting a separation speed.
The CIEF-HF FIFFF [D. Kang, M. Moon, Anal. Chem., 2006, 78, pp. 5789-5798] has an advantage in that the proteins can be separated in two dimensions without using the gel, and a disadvantage in that an amount of separable proteins has no choice but is restricted due to a limitation of a capacity of the capillary in the event of the primary isoelectric focusing the capillary. Further, while a fraction of the proteins separated according to pI after the primary separation is transferred to a hollow fiber module, and then separated according to molecular weight, the other proteins stand by in the capillaries, and then are separated in the hollow fiber module. For this reason, a total separation time is prolonged by a desired fraction of pI. In addition, while some of the protein samples primarily separated according to pI are secondarily separated in the hollow fiber module according to the molecular weight, the other pI fractions must stand by in the capillaries under the electric field. In this process, the proteins are slightly shifted due to the influence of an electroosmotic flow driven on inner walls of the capillaries. This results in a problem that allows the separated fractions to be mixed again, and causes contamination of the fractions. In the case of the CIEF-HF FIFFF, because a maximum amount of the proteins that can be injected at once is about 40 μg, there is the limitation of a capacity to some extents to process a large amount of proteome samples.